optical-photothermal-infrared-fluorescence-situ-hybridization-optir

Optical Photothermal Infrared-Fluorescence In Situ Hybridization (OPTIR-FISH)

Understanding the metabolic activities of individual cells within complex communities is critical for unraveling their role in human disease. Here, we ...

Boston University

We report rapid and sensitive phenotyping of bacterial response to antibiotic treatment at single-cell resolution by a Raman-integrated optical mid-infrared photothermal (MIP) microscope. The MIP microscope successfully detected biochemical changes of bacteria in specific to the acting mechanism of erythromycin with 1 h incubation. Compared to Raman spectroscopy, MIP spectroscopy showed a much larger signal-to-noise ratio at the fingerprint region at an acquisition speed as fast as 1 s per spectrum. The high sensitivity of MIP enabled detection of metabolic changes at antibiotic concentrations below minimum inhibitory concentration (MIC). Meanwhile, the single-cell resolution of the technique allowed observation of heteroresistance within one bacterial population, which is of great clinical relevance. This study showcases characterizing antibiotic response as one of the many possibilities of applying MIP microscopy to single-cell biology.

Fingerprinting Bacterial Metabolic Response to Erythromycin by Raman-Integrated Mid-Infrared Photothermal Microscopy.

Midinfrared photothermal (MIP) microscopy, also called optical photothermal infrared (O-PTIR) microscopy, is an emerging tool for bond-selective chemical imaging of living biological and material samples. In MIP microscopy, a visible probe beam detects the photothermal-based contrast induced by a vibrational absorption. With submicron spatial resolution, high spectral fidelity, and reduced water absorption background, MIP microscopy has overcome the limitations in infrared chemical imaging methods. In this review, we summarize the basic principle of MIP microscopy, the different origins of MIP contrasts, and recent technology development that pushed the resolution, speed, and sensitivity of MIP imaging to a new stage. We further emphasize its broad applications in life science and material characterization, and provide a perspective of future technical advances.

Mid-Infrared Photothermal Microscopy: Principle, Instrumentation, and Applications.

Antimicrobial resistance poses great threats to global health and economics. Current gold-standard antimicrobial susceptibility testing (AST) requires extensive culture time (36-72 h) to determine susceptibility. There is an urgent need for rapid AST methods to slow down antimicrobial resistance. Here, we present a rapid AST method based on wide-field mid-infrared photothermal imaging of protein synthesis from C-glucose in . Our wide-field approach achieved metabolic imaging for hundreds of bacteria at the single-cell resolution within seconds. The perturbed microbial protein synthesis can be probed within 1 h after antibiotic treatment in  cells. The susceptibility of antibiotics with various mechanisms of action has been probed through monitoring protein synthesis, which promises great potential of the proposed platform toward clinical translation.

High-Throughput Antimicrobial Susceptibility Testing of  by Wide-Field Mid-Infrared Photothermal Imaging of Protein Synthesis.

Simultaneous identification and metabolic analysis of microbes with single-cell resolution and high throughput are necessary to answer the question of "who eats what, when, and where" in complex microbial communities. Here, we present a mid-infrared photothermal-fluorescence in situ hybridization (MIP-FISH) platform that enables direct bridging of genotype and phenotype. Through multiple improvements of MIP imaging, the sensitive detection of isotopically labeled compounds incorporated into proteins of individual bacterial cells became possible, while simultaneous detection of FISH labeling with rRNA-targeted probes enabled the identification of the analyzed cells. In proof-of-concept experiments, we showed that the clear spectral red shift in the protein amide I region due to incorporation of C atoms originating from C-labeled glucose can be exploited by MIP-FISH to discriminate and identify C-labeled bacterial cells within a complex human gut microbiome sample. The presented methods open new opportunities for single-cell structure-function analyses for microbiology.

Mid-Infrared Photothermal-Fluorescence In Situ Hybridization for Functional Analysis and Genetic Identification of Single Cells.

By optically sensing absorption-induced photothermal effect, mid-infrared (IR) photothermal (MIP) microscope enables super-resolution IR imaging of biological systems in water. However, the speed of current sample-scanning MIP system is limited to milliseconds per pixel, which is insufficient for capturing living dynamics. By detecting the transient photothermal signal induced by a single IR pulse through fast digitization, we report a laser-scanning MIP microscope that increases the imaging speed by three orders of magnitude. To realize single-pulse photothermal detection, we use synchronized galvo scanning of both mid-IR and probe beams to achieve an imaging line rate of more than 2 kilohertz. With video-rate speed, we observed the dynamics of various biomolecules in living organisms at multiple scales. Furthermore, by using hyperspectral imaging, we chemically dissected the layered ultrastructure of fungal cell wall. Last, with a uniform field of view more than 200 by 200 square micrometer, we mapped fat storage in free-moving  and live embryos.

Video-rate mid-infrared photothermal imaging by single-pulse photothermal detection per pixel.

Three-dimensional molecular imaging of living organisms and cells plays a significant role in modern biology. Yet, current volumetric imaging modalities are largely fluorescence-based and thus lack chemical content information. Mid-infrared photothermal microscopy as a chemical imaging technology provides infrared spectroscopic information at submicrometer spatial resolution. Here, by harnessing thermosensitive fluorescent dyes to sense the mid-infrared photothermal effect, we demonstrate 3D fluorescence-detected mid-infrared photothermal Fourier light field (FMIP-FLF) microscopy at the speed of 8 volumes per second and submicron spatial resolution. Protein contents in bacteria and lipid droplets in living pancreatic cancer cells are visualized. Altered lipid metabolism in drug-resistant pancreatic cancer cells is observed with the FMIP-FLF microscope.

3D Chemical Imaging by Fluorescence-detected Mid-Infrared Photothermal Fourier Light Field Microscopy.

Clinical identification and fundamental study of viruses rely on the detection of viral proteins or viral nucleic acids. Yet, amplification-based and antigen-based methods are not able to provide precise compositional information of individual virions due to small particle size and low-abundance chemical contents (e.g., ~ 5000 proteins in a vesicular stomatitis virus). Here, we report a widefield interferometric defocus-enhanced mid-infrared photothermal (WIDE-MIP) microscope for high-throughput fingerprinting of single viruses. With the identification of feature absorption peaks, WIDE-MIP reveals the contents of viral proteins and nucleic acids in single DNA vaccinia viruses and RNA vesicular stomatitis viruses. Different nucleic acid signatures of thymine and uracil residue vibrations are obtained to differentiate DNA and RNA viruses. WIDE-MIP imaging further reveals an enriched β sheet components in DNA varicella-zoster virus proteins. Together, these advances open a new avenue for compositional analysis of viral vectors and elucidating protein function in an assembled virion.

Article	Year
Fingerprinting Bacterial Metabolic Response to Erythromycin by Raman-Integrated Mid-Infrared Photothermal Microscopy. Analytical chemistry\| PubMed ID: 33089997	2020
Mid-Infrared Photothermal Microscopy: Principle, Instrumentation, and Applications. The journal of physical chemistry. B\| PubMed ID: 36285985	2022
High-Throughput Antimicrobial Susceptibility Testing of by Wide-Field Mid-Infrared Photothermal Imaging of Protein Synthesis. Analytical chemistry\| PubMed ID: 36651850	2023
Mid-Infrared Photothermal-Fluorescence In Situ Hybridization for Functional Analysis and Genetic Identification of Single Cells. Analytical chemistry\| PubMed ID: 36652555	2023
Video-rate mid-infrared photothermal imaging by single-pulse photothermal detection per pixel. Science advances\| PubMed ID: 37315131	2023
3D Chemical Imaging by Fluorescence-detected Mid-Infrared Photothermal Fourier Light Field Microscopy. Chemical & biomedical imaging\| PubMed ID: 37388959	2023
Single virus fingerprinting by widefield interferometric defocus-enhanced mid-infrared photothermal microscopy. Nature communications\| PubMed ID: 37863905	2023

Zhongyue Guo

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